Fibronectin receptor

ABSTRACT

The present invention provides a substantially pure integrin-type receptor characterized in that it consists of an  alpha v and a  beta 1 subunit. The  alpha v beta 1 integrin binds to fibronectin and GRGDSPK but does not bind to vitronectin. The  alpha v beta 1 integrin can be used to determine the presence of a  alpha v beta 1 ligand and to develop adhesion peptides specific for the various integrins. The presence of the  alpha v beta 1 integrin can be used to assess ability of cells to adhere to fibronectin.

BACKGROUND OF THE INVENTION

This invention relates to receptors for adhesion peptides, and morespecifically to a novel receptor having affinity for fibronectin.

Multicellular organisms, such as man, have some 10¹⁴ cells which can bedivided into a minimum of fifty different types, such as blood cells andnerve cells. During the course of growth and development, cells adhereto other cells, or to extracellular materials, in specific and orderlyways. Such cell adhesion mechanisms appear to be of importance inmediating patterns of cellular growth, migration and differentiation,whereby cells develop specialized characteristics so as to function as,for example, muscle cells or liver cells. Cell adhesion mechanisms arealso implicated in dedifferentiation and invasion, notably where cellslose their specialized forms and become metastasizing cancer cells.

The mechanisms underlying the interactions of cells with one another andwith extracellular matrices are not fully understood, but it is thoughtthat they are mediated by cell surface receptors which specificallyrecognize and bind to a cognate ligand on the surface of cells or in theextracellular matrix.

The adhesion of cells to extracellular matrices and their migration onthe matrices is mediated in many cases by the binding of a cell surfacereceptor to an Arg-Gly-Asp containing sequence in the matrix protein (asreviewed in Ruoslahti and Pierschbacher, Science 238:491(1987)). TheArg-Gly-Asp sequence is a cell attachment site at least in fibronectin,vitronectin, various collagens, laminin and tenascin. Despite thesimilarity of their cell attachment sites, these proteins can berecognized individually by the specific receptors.

Integrins are a family of adhesion receptors which bind to Arg-Gly-Aspbinding sites of extracellular matrix membrane proteins via theArg-Gly-Asp binding sites. They are heterodimeric molecules composed ofone alpha (α) and one beta (β) subunit. Several subunits of each kindare known, and various αβ combinations make up receptors with differentligand specificities.

Eleven distinct alpha chains have thus far been described. Formerly,they have been divided into three main subfamilies based on the betasubunit with which they associate. The β₁ subfamily includes receptorsfor fibronectin, various collagens, laminin and tenascin. The β₂subfamily consists of leukocyte specific receptors, while the β₃subfamily contains multispecific receptors commonly referred to as theplatelet glycoprotein IIb-IIIa and the vitronectin receptor. Among thecombinations known to exist, the α_(V) subunit associates with the β₃subunit to form a vitronectin receptor and with two recently described βsubunits called β_(X) and β_(S). The α_(V) β_(X) integrin is avitronectin and fibronectin receptor while the ligand specificity ofα_(V) β_(S) is not known.

Because of the importance of integrins in mediating critical aspects ofboth normal and abnormal cell processes, there exists the need toidentify and characterize different integrins. The present inventionsatisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

The present invention provides a substantially pure integrin-typereceptor characterized in that it consists of an α_(V) and a β₁ subunit.The α_(V) β₁ integrin binds to fibronectin and GRGDSPK but does not bindto vitronectin. The α_(V) β₁ integrin can be used to determine thepresence of a α_(V) β₁ ligand and to develop adhesion peptides specificfor the various integrins. The presence of the α_(V) β₁ can be used toassess ability of cells to adhere to fibronectin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph of a gel showing integrin subunits expressed onvarious cell types.

FIG. 2 shows the results of cell adhesion assays on fibronectin andvitronectin. The error bars indicate the standard error of the mean ofthree independent assays.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a new receptor composed of an α_(V) and a β₁subunit, or their immunological equivalents. This integrin-type receptoris herein termed "α_(V) β₁ receptor" or "α_(V) β₁ integrin." The α_(V)β₁ receptor is immunoprecipitated with a monoclonal antibody to theα_(V) subunit and includes a band in the expected position of the β₁subunit, as shown in FIG. 1, left panel.

To confirm the association between the α_(V) and β₁ subunits implied bythe immunoprecipitation results described above, monoclonal antibodiesto each of the subunits were used to isolate receptor complexes from thefibroblast cell line WI-38. A series of antibodies were then used toidentify the co-isolated subunit. The material purified by theanti-α_(V) monoclonal antibody was precipitated by two different anti-β₁monoclonal antibodies and by a polyclonal serum to a peptiderepresenting the β₁ cytoplasmic domain. All three anti-β₁ reagentsrecognized the α_(V) -containing integrin. Conversely, the materialobtained with a β₁ monoclonal antibody was immunoprecipitated by twodifferent anti-α_(V) monoclonal antibodies and by a polyclonal serum toa peptide representing the α_(V) cytoplasmic domain. These data indicatethat the α_(V) and β₁ subunits do associate to form a complex.

To investigate the ligand binding specificity of the new α_(V) β₁integrin, affinity chromatography experiments and cell adhesion assayswere performed. In the chromatography experiments, detergent extracts ofIMR 32 neuroblastoma cells surface labelled with ¹²⁵ I were fractionatedon fibronectin and GRGDSPK peptide affinity columns. The α_(V) β₁integrin bound to a 110 Kd fragment of fibronectin that contains thecell attachment site. It was eluted from the column with a peptide(GRGDSP), that represents the cell attachment site, but not a relatedpeptide, GRGESP. No additional bands appeared with subsequent EDTAelution. The receptor also bound to a column containing the peptideGRGDSPK coupled to Sepharose and was eluted with the GRGDSP peptide butnot with the GRGESP peptide.

Amino acids are identified herein by the standard one-letterabbreviations, as follows:

    ______________________________________                                        Amino Acid     Symbol                                                         ______________________________________                                        Alanine        A                                                              Aspartic acid  D                                                              Cysteine       C                                                              Glutamine      Q                                                              Glutamic acid  E                                                              Glycine        G                                                              Histidine      H                                                              Isoleucine     I                                                              Leucine        L                                                              Lysine         K                                                              Methionine     M                                                              Phenylalanine  F                                                              Proline        P                                                              Serine         S                                                              Threonine      T                                                              Tryptophan     W                                                              Tyrosine       Y                                                              Valine         V                                                              ______________________________________                                    

To confirm that the eluted material was the α_(V) β₁ complex, the peakfractions from each column were pooled and immunoprecipitated withmonoclonal antibodies to the α_(V) and β₁ subunits. Both antibodiesprecipitated the same two bands from each column, indicating that thematerial specifically eluted from each column was, in fact, α_(V) β₁.

Cell attachment assays showed that the IMR 32 cells attached tofibronectin but not to vitronectin (FIG. 2) or fibrinogen (not shown).The adhesion of these cells to fibronectin appears to be mediated by theα_(V) β₁ complex since the only other integrin detected, α₁ β₁, did notbind fibronectin in the affinity chromatography experiments (see FIG.1). These cells also attached to collagens I and IV and lamininpresumably due to the presence of the α₁ β₁ complex.

The data indicate that the α_(V) β₁ integrin subunits associate to forma functional fibronectin receptor. Although molecular heterogeneity dueto modifications such as alternative splicing cannot be entirely ruledout, the subunits of the new receptor were immunologicallyindistinguishable from α_(V) and β₁ with at least three antibodies toeach subunit. Therefore, by the criteria of electrophoretic mobility andimmunological reactivity, the new receptor is composed of α_(V) and β₁subunits or their immunological equivalents.

The new α_(V) β₁ does not bind to vitronectin but can be isolated on aGRGDSPK column. This ligand binding pattern appears to be different fromthat of any of the previously characterized integrins. The ability ofthis receptor to bind to a GRGDSPK column is a property shared by twovitronectin-binding integrins, α_(V) β₃ (Pytela, et al., Proc. Natl.Acad. Sci. USA 82:5766 (1985)), and the platelet receptor α_(IIB) β₃(Pytela, et al., Science 231:1559 (1986) (and references therein), whichare incorporated herein by reference). A complex between α_(V) and therecently described β_(S) subunit may also belong to this group (Freed,et al., EMBO 8:2955 (1989), which is incorporated herein by reference).Another recently described complex of α_(V) (α_(V) β_(X)) binds to bothfibronectin and vitronectin (Cheresh et al., Cell 57:59 (1989), which isincorporated herein by reference).

Fibronectin-binding integrins of the β₁ class (α₅ β₁) do not bind tovitronectin and unlike the α_(V) β₁ integrin described here, do not binddetectably to the GRGDSPK column. Therefore, the α_(V) β₁ complexappears to have a distinct intermediate specificity between thevitronectin binding integrins and the β₁ class integrins.

Three different α subunits have been shown to associate with more thanone β subunit. Two of these, α₄ and α₆, can pair with either one of twoβ subunits. The α_(V) subunit appears to be especially versatile sinceit has already been shown to be capable of associating with four βsubunits. Moreover, the association between α_(V) and β₁ described hereunexpectedly crosses the boundaries of two previously proposed integrinclasses, forcing a reevaluation of the currently accepted integrinclassification.

Since receptors for collagens, laminin and fibronectin all share acommon β subunit, it has been proposed that the α subunit determines thespecificity of integrins. The new α_(V) β₁ integrin described here is afibronectin receptor, whereas α_(V) β₃ is a vitronectin receptor. Thisresult, along with the demonstration that α_(V) β_(X) binds tofibronectin, shows that the β subunit plays a greater role indetermining receptor specificity than thought previously.

The α_(V) β₁ is useful in assaying the ability of cells to attach toextracellular matrices; the presence of α_(V) β₁ on the cell surfaceindicates the ability to attach to fibronectin. The presence of theα_(V) β₁ integrin is detected in an immunoassay format using an antibodyagainst each of the subunits as described in Example I or by amodification of that immunoassay format. Such assays are well known tothose skilled in the art. See generally, ANTIBODIES; A LABORATORY MANUAL(Harlow and Lane, eds.) Cold Spring Harbor Laboratory (1988), which isincorporated herein by reference.

Another area where the α_(V) β₁ receptor is useful is the analysis ofligands for integrins. The specificity of such ligands is important. Forexample, synthetic peptides containing the RGD sequence that bind to theplatelet integrin gp IIb/IIIa but not to other integrins are beingdeveloped into anti-platelet pharmaceuticals.

The ability of a compound to interact with the α_(V) β₁ integrin can beassessed by affinity chromatography as described under Example II. Acell attachment assay can be used as described under Example III whenthe contribution by other integrins possessed by the test cells can beexcluded. Finally an enzyme immunoassay format or a radioreceptor assaycan be used as described in Hautanen et al., J. Biol. Chem.264:1347-1442 (1989); Gehlsen et al., J. Biol. Chem., in press (1989).

The following examples are intended to illustrate but not limit theinvention.

EXAMPLE I Identification of α_(V) β₁ Integrin

Antibodies to the integrin subunits were prepared as indicated in thefollowing table:

                  TABLE I                                                         ______________________________________                                                     monoclonal           reference                                   sub-         or                   or                                          unit host    polyclonal                                                                              immunogen  confirmation                                ______________________________________                                        α.sub.v                                                                      mouse   monoclonal                                                                              purified   immunoblotting;                                          Mab 147   vitronectin                                                                              reactive with                                                      receptor   α.sub.v subunit                       α.sub.v                                                                      mouse   Mab 59    purified   immunoblotting;                                                    vitronectin                                                                              reactive with                                                      receptor   α.sub.v subunit                       α.sub.v                                                                      rabbit  polyclonal                                                                              KRVRPPQEE- Freed et al.                                                       QEREQLQPH- EMBO J.                                                            ENGEGNSET  8:2955 (1989)                               α.sub.5                                                                      rabbit  polyclonal                                                                              EKAQLKP-   immunoblotting;                                                    PATSDA     reactive with                                                                 α.sub.5 subunit                       α.sub.6                                                                      mouse   monoclonal                                                                              α.sub.6                                                                            Sonneberg et al.,                                        GoH3                 J. Biol. Chem.                                                                263:14030 (1988)                            α.sub.2                                                                      mouse   monoclonal                                                                              α.sub.2                                                                            Wagner and                                               PIH5                 Carter                                                                        J. Cell Biol.                                                                 105:1073 (1987)                             α.sub.3                                                                      rabbit  polyclonal                                                                              cytoplasmic                                                                              Hynes et al.,                                                      domain of  J. Cell Biol.                                                      α.sub.3 subunit                                                                    109:409 (1989)                              β.sub.1                                                                       rabbit  polyclonal                                                                              KKKEKEKMN- immunoblotting;                                                    AKWDTGENP- reactive with                                                      IYSAVTTVV- β.sub.1 subunit                                               NPKYEGK                                                β.sub.1                                                                       mouse   monoclonal                                                                              purified   immunoblotting;                                          LM 534    fibronectin                                                                              reactive with                                            LM 442    receptor   β.sub.1 subunit                        ______________________________________                                    

Human neuroblastoma cells (IMR 32; ATCC Accession No. CCL 127), lungcell fibroblasts (WI-38; ATCC Accession No. CCL 75), for example,(WI-38; ATCC Accession No. CCL 757) and glioblastoma cells (U251) weresurface labeled with ¹²⁵ I and lactoperoxidase according to the methodof Pytela et al., Cell 40:191-198 (1985), which is incorporated hereinby reference, and extracted with a buffer containing a 0.5%Triton-X-100, 150 mM NaCl, 1 μg/ml leupeptin, 1 mg/ml aprotinin, 0.4μg/ml pepstatin and 10 mM Tris, pH 7.2. Integrin heterodimers wereimmunoprecipitated with antibodies specific for either the β₁ or αsubunits and analyzed by SDS-PAGE. Briefly, the extracts were clarifiedat 15,000 rpm and precleared by an incubation with preimmune rabbit ormouse IgG-Sepharose. Following an incubation with the primaryantibodies, immunocomplexes were uncovered with either Sepharose-ProteinA or Sepharose-goat anti-mouse IgG.

α_(V) -containing integrins and β₁ -containing integrins wereimmunopurified from the WI-38 extract on anti-α_(V) (Mab 147) andanti-β₁ (Mab LM 534) Sepharose columns respectively. The column waseluted with 50 mM glycine-HCl pH 3, containing 0.5% Triton-X-100. Afterneutralization, the material was divided in three aliquots forimmunoprecipitation with anti-β₁ antibodies or anti-α_(V) antibodies andthe immunoprecipitates were analyzed by SDS-PAGE substantially asdescribed above. In each case association between the α_(V) and the β₁subunits was found.

EXAMPLE II Analysis of Ligand Specificity and Purification of α_(V) β₁Integrins

IMR 32 cells were surface labeled with ¹²⁵ I and lysed in 200 mMoctylglucoside, 150 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, 1 mM MnCl₂, 1 μg/mlleupeptin, 1 μg/ml aprotinin, 0.4 μg/ml pepstatin, and 10 mM Tris, pH7.2. The cell extract was applied to a 110 kD fibronectinfragment-Sepharose column and the column was washed with 50 mMoctylglucoside, 1 mM CaCl₂, 1 mM MgCl₂, 1 mM MnCl₂, 150 mM NaCl, and 10mM Tris, pH 7.2, alone and with 1 mg/ml GRGESP peptide. The column wassubsequently eluted with 1 mg/ml GRGDSP peptide followed by elution with10 mM EDTA. IMR 32 cell extract was also fractionated by identical meanson a GRGDSPK column.

These procedures were similar to those described in Pytela et al., Meth.Enzymol. 144:475-489 (1987); and Gailit and Ruoslahti, J. Biol. Chem.263:12927-12932 (1988), which are incorporated herein by reference. Theeluates from each of these columns contained an integrin with an α and aβ subunit. The peak fractions were pooled and immunoprecipitated withthe anti-α_(V) (Mab 147), or anti-β₁ (Mab LM 534) described in ExampleI. The integrin bound to the column was found to precipitate with bothanti-α_(V) and anti-β₁, indicating that it is an association of theα_(V) and β₁ subunits.

EXAMPLE III Cell Adhesion Assays

Microtiter plates were coated with various concentrations of fibronectinand vitronectin and postcoated with 0.05% bovine serum albumin. Afterwashing, approximately 10⁵ IMR 32 (human neuroblastoma; ATCC CCl 127) orMG-63 (human osteosarcoma; ATCC CCL 1427) cells were plated per well andincubated at 37° C. for 90 minutes. The attached cells were fixed in 3%paraformaldehyde and stained with 0.5% crystal violet. The attachmentwas quantitated by reading the absorbance at 600 nm. As shown in FIG. 2,the IMR 32 cells attach to fibronectin, but not to vitronectin orfibrinogen, whereas the MG-63 cells attach to all three substrates.

Although the invention has been described with reference to thepresently-preferred embodiment, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

We claim:
 1. A substantially pure active integrin comprising subunitsα_(V) and β₁ or their immunological equivalents, wherein said integrinbinds to GRGDSPK in a cell adhesion assay.